Impaired axonal transport may play an early, pivotal role in a variety of neurodegenerative disorders including Parkinson's disease (PD). When axon transport is disrupted, trophic support is lost, synaptic vesicles and transmitters are depleted, biological materials accumulate, degeneration occurs and ultimately the neuron dies. As a pathological precursor, impaired axonal transport is particularly compelling in PD because disruption of synaptic function and the presence of axon pathology is an early, common feature. Research in this area is limited, however, by the lack of tools to selectively image and target CNS axons. Towards this end, we have modified a compartmented system designed for peripheral cultures such that CNS neurons can be grown with axons segregated to one side of a barrier. When used with GFP-labeled dopaminergic neurons derived from genetically engineered mice, dopaminergic axons can be examined using live cell, real time imaging. The goal of the current application is to utilize our unique ability to monitor axonal transport in CNS dopaminergic neurons to test a nested set of hypotheses that PD-associated neurotoxins and genetic mutations trigger early changes in axon transport that contribute to the loss of synaptic function and cell death. Specifically, we hypothesize that the PD-mimetic MPP+ affects mitochondrial and vesicular axon trafficking leading to the loss of synaptic function, that this occurs via mitochondrial-dependent and independent processes, and that mutations in the PD-linked gene LRRK2 will affect similar transport processes of organelles and vesicles as MPP+. Optical, molecular and cellular techniques will be used to determine axonal transport and signaling pathways associated with mitochondrial and vesicular movement in dopaminergic axons. These experiments will provide insights impossible to obtain from standard culture or animal models. Taken together, the proposed studies will determine whether environmentally or genetically induced axonal injury plays a central role in the death of dopaminergic neurons. If so, novel therapies can be targeted to the elucidated control points in order to stop or slow disease progression. PUBLIC HEALTH RELEVANCE Environmental or genetic factors associated with Parkinson's disease may affect fundamental mechanisms underlying the way in which a neuron sends biological materials down an axon. This may compromise the ability of neurons to communicate with each other, lead to the accumulation of proteins and organelles, and ultimately cause the axon and subsequently the neuron to degenerate. Identifying and characterizing how axonal injury occurs in Parkinson's disease has enormous potential for the discovery of novel therapies that can be targeted to the elucidated control points in order to stop or slow disease progression.